L-alanine is the most abundant amino acid found in E. coli and is the major metabolite for gluconeogenesis in mammals. The overall aim of our research is to determine, and then analyze, the routes of L-alanine biosynthesis, both genetically and physiologically. We will utilize methodologies similar to those which we have employed in our studies on the organization and control of the isoleucine-valine biosynthetic genes and their corresponding enzymes in Escherichia coli K-12 and Salmonella typhimurium LT2. We plan to determine the routes of L-alanine biosynthesis by the isolation and characterization of transposon-induced mutations blocked in each of the alternate biosynthetic routes as well as in different steps in the same route. The regulation of the genes involved in L-alanine biosynthesis and of the enzymes they encode will be studied by performing appropriate enzyme assays on cells grown under different physiological conditions and by performing the assays in the presence of different potential inhibitors. The effect of gene dosage upon the expression of the single chromosomal copy of the gene will be assayed in strains carrying a lac fusion allele on the chromosome and the wildtype allele on a multicopy plasmid. These studies will elucidate the biosynthesis of the last remaining amino acid whose metabolism has not been subjected to extensive genetic, physiological and/or biochemical scrutiny.